The long term goal of the studies proposed is to improve our understanding at the molecular level of the membrane receptors involved in recruitment of circulating monocytes to the developing atherosclerotic lesion and the conversion of those monocytes to foam cells. Emphasis will be placed on MCP-1 and its receptor, CCR2, because the central role of this chemokine pair early lesion formation has been clearly established by gene targeting experiments. We have previously demonstrated that CCR2 expression in monocytes is reduced by ligands for the nuclear receptor PPAR. We propose intensive studies of this and other methods of regulating the expression of CCR2 and also the regulation of other chemokine receptors and adhesion molecules, including CX3CR1, CDlla, CD1lb and CD49d. We have recently developed a novel approach to quantifying the recruitment of monocytes to the arterial wall in vivo in mouse models of atherosclerosis. This will be applied to studying the role of MCP-1 and CCR2 quantitatively by comparing wild-type mice with mice deficient in CCR2 or MCP-1. Using this new method we will also test the hypothesis that monocyte recruitment into developing lesions is preceded by a build-up of oxidized LDL in the sub-endothelial space. Another approach to this question will make use of mice deficient in 12/15-lipoxygenase, an enzyme believed to be involved in LDL oxidation. The role of CCR2 and of adhesion molecules will be studied clinically to determine whether regulation in humans parallels that previously described in mice. In these studies we will also test the reversibility of the effects of hypercholesterolemia on monocyte gene expression. Finally we will study the structure and function of receptors involved in the binding of oxidized LDL and of apoptotic cells, with emphasis on CD36 and CD68. We have shown that CD36 can bind either the lipid moiety of oxidized LDL or its isolated apoprotein B. Whether the binding occurs at the same sites or different sites remains to be determined. This will be approached by site-directed mutagenesis and by the preparation of chimeric receptors. Additionally, we will explore some apparent anomalies with regard to the ability of CD36 to internalize and cause degradation of oxidized LDL. Recently Dr. J. Aldon Lusis at UCLA, has succeeded in partially knocking out the gene for CD68. In collaboration with Dr. Lusis, we propose to test further the possible role of CD68 in the recognition and uptake of oxidized LDL and apoptotic cells. Finally, in collaboration with Dr. Jonathan Tait at the University of Washington, we will evaluate the ligand-binding properties of recombinant CD68 made in E. coli and therefore deficient in glycosyl post-translational additions.